Incidence, risk factors, and clinical findings of syphilis among men living with HIV in Croatia during the COVID-19 pandemic

We conducted a nationwide longitudinal observational study to estimate the incidence of syphilis in a cohort of male persons living with HIV (MLWH) in Croatia in the pre-COVID-19 and COVID-19 years. Data were reviewed and extracted from the clinical database. We analyzed 1187 MLWH (≥ 18 years) in care in Croatia from 2018 to 2021 and used Poisson regression to calculate rates. We observed a 91.4% increase in incidence between 2019 and 2020; the overall rate was 6.0/100 person-years, and the annual rate ranged from 3.3/100 person-years in 2018 to 9.3/100 person-years in 2021. We found higher rates in men who have sex with men, MLWH with a baseline history of syphilis, MLWH with a more recent HIV diagnosis, and a lower rate in those who had clinical AIDS. The rate of syphilis serological testing was 3.5% lower in 2020 compared to 2019. Recurrent syphilis was more likely asymptomatic compared to the first episodes. In conclusion, during the COVID-19 epidemic years, there was a huge increase in syphilis. Results highlight the need for enhanced and novel prevention interventions.

Soon after the first reported case of the coronavirus disease (COVID-19) caused by a new strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 in China, the world faced a global health crisis. Considering all preventive measures to control the spread of SARS-CoV-2 infection through quarantine and social distancing, it was to be expected that there would be changes in sexual behaviors leading to a decrease in the incidence of sexually transmitted diseases (STDs). However, the data on STDs during the COVID-19 epidemic years is still evolving and the impact of the COVID-19 pandemic on STDs remains to be defined and might be different in different countries. Some studies reported a decrease in the number of reported STDs particularly during the lockdown periods [1][2][3][4][5][6] , whereas other studies found no significant change 7,8 or an increase in the number of reported STDs, including syphilis [9][10][11][12][13][14][15][16] .
Syphilis is a highly contagious bacterial infection caused by gram-negative spirochaete Treponema pallidum and is typically spread through sexual contact. The World Health Organization estimated that seven million people got infected with syphilis worldwide in 2020 17 , the highest incidence was recorded among men who have sex with men (MSM) 18 .
In the most recent European Centre for Disease Prevention and Control (ECDC) surveillance report 35,039 confirmed syphilis cases were reported in 29 EU/EEA Member States, with a crude notification rate of 7.4 cases per 100,000 population 19 . The majority of cases (73%) were reported in MSM, and the trends in MSM showed a steep increase in reported cases from 2010 up to 2017, with a much slower increase between 2018 and 2019 19 . Syphilis is a notifiable disease in Croatia, however, because of underreporting of cases the notification reports from the Croatian Nation Public Health Institute do not seem reliable. In the ECDC report the annual number of reported cases of syphilis ranged from 25 in 2015 to 35 in 2018 in Croatia. There have been two studies that estimated the prevalence of past syphilis among MSM measured by Treponema pallidum hemagglutination in Croatia. Both studies used a respondent-driven sampling method and reported a prevalence of syphilis of 10.6% in 2006 and 7.6% in 2011 20 . The aim of this study was to estimate the incidence of syphilis diagnoses in a cohort of male persons living with HIV (MLWH) in Croatia in the pre-COVID-19 and COVID-19 years and to identify syphilis risk factors in this population.

Methods
Setting. In Croatia, all people living with HIV (PLWH) are treated at the University Hospital for Infectious Diseases (UHID) in Zagreb. The HIV Outpatient Department at UHID has been established in 1997, it currently provides outpatient care for PLWH including treatment for common STDs such as gonorrhea, chlamydia, and syphilis. Antiretroviral therapy is also given from the hospital pharmacy. Because of the distance from the residence of PLWH to the treatment center, phone consultations were part of routine care since 1997 and a mHealth solution delivering laboratory results to the smartphones of PLWH has been introduced in 2016 21 . Antiretroviral therapy (ART) can also be delivered to the home address, this is usually done after a phone consultation.
Serologic testing for syphilis at UHID is usually routinely done annually or bi-annually, however, it may be done more or less frequently depending on the clinical findings and sexual behavior history. Since 2004, the testing algorithm for syphilis screening includes first a treponemal test (the Treponema pallidum hemagglutination assay [TPHA]), followed by a nontreponemal test (the Rapid Plasma Regain [RPR] test).
Population and definitions. Because no women had syphilis, we restricted our analysis to men (Fig. 1) and since there were only 3 transgender women, we refer to men as males at birth. Included were men (> 18 years) living with HIV receiving care at UHID with at least 1 year of follow-up (n = 1187, Fig. 1). The observation period started on or after January 1st, 2018, and ended on December 31st, 2021, or earlier if MLWH died, moved, or were lost to follow-up. The person's data, including demographic characteristics, diagnoses, prescribed medications, number of syphilis tests, and laboratory values, were reviewed and extracted from the electronic database. Diagnoses of primary, secondary, early latent (infection within 1 year) and late latent syphilis (infection > 1 year), and neurosyphilis were made by clinicians at UHID. A syphilis clinical diagnosis typically included a history of exposure, physical findings (e.g. ulcers, rash, lymphadenopathy, oral mucosal erosions, etc.), and serological tests.
Incident syphilis was defined as a new syphilis diagnosis, it included both first-time and reinfection events. We defined incident cases of syphilis based on clinical diagnosis, prescribed treatment, and serological testing. Syphilis was considered present if any of the following were observed: both a documented syphilis clinical diagnosis and treatment for syphilis, a positive syphilis TPHA screen when a previous test was negative, and a four-fold increase in RPR titer. Episodes of syphilis at inclusion into the study (n = 18) were not considered incident cases. Past history of syphilis at inclusion into care was defined if treatment was recalled by the patient or the TPHA test was positive.
The Ethics Committee of the University Hospital for Infectious Diseases (UHID), Zagreb, Croatia approved the study (No. 01-1931(No. 01- -7-2020 www.nature.com/scientificreports/ continuous variables. The chi-square trend test was used to examine the proportion of asymptomatic syphilis cases from 2018 to 2021. We also examined whether the first syphilis episode after inclusion into care was more likely asymptomatic in MLWH with a baseline history of past or current syphilis. Using Poisson regression, we calculated crude incidence rates for incident syphilis (first and repeated episodes) by sociodemographic and clinical characteristics. To examine factors related to syphilis diagnoses, incident rate ratios with 95% confidence intervals (CI) were calculated. The calendar year was annualized, and we used a generalized estimating equations (GEE) model with an exchangeable correlation structure to account for recurrent events. Age, viral load, prior episode of syphilis, clinical AIDS, and hepatitis C infection on follow-up were time-updated variables whereas other variables were fixed. We modeled the total number of events using an offset for the person-year follow-up time. Crude analyses included the outcome and one predictor variable whereas multivariable analyses included more than one predictor. Statistical analyses were done using SAS version 9.4 (SAS Institute, Inc., Cary, North Carolina). P-values were two-sided and statistical significance was set at the conventional P-value of < 0.05. Ethical approval. The study was approved by the University Hospital of Infectious Diseases Ethics Committee.

Results
Descriptive analysis. Of 1187 males at birth, three were transgender women and all, except one man, were Caucasians. The HIV transmission risk was: sex between men (n = 976, 82.2%), heterosexual (n = 142, 12.0%), injection drug use (n = 28, 2.4%), recipient of blood or blood products (n = 5, 0.4%), mother to child transmission (n = 4, 0.3%) and unknown (n = 32, 2.7%). At baseline, the median age was lower among MLWH who had incident syphilis compared to those who did not (40.4 years versus 42.0 years) ( Table 1). Median age and different age categories at a syphilis diagnosis on follow-up are presented in the Supplementary Material. MLWH who had syphilis on follow-up were more likely MSM, living in Zagreb, diagnosed with HIV in the period 2016 to 2020, and did not have clinical AIDS (Table 1). The median CD4 lymphocyte count at baseline was high (599.5 per mm 3 ), 75.2% had < 50 copies of HIV1-RNA per ml, and 91.8% were ART-experienced (Table 1) Of 254 syphilis events, 116 (45.7%) were the first-ever diagnoses. The incidence rate for first-ever syphilis events was 5.0 (95% CI, 4.2-5.9) per 100 PYFU, and for the recurrent event, it was 8.3 (95%, 6.8-10.0) per 100 PYFU. There was an increase in the incidence of syphilis in the period 2018-2021 (Table 2, Fig. 2A). This increase was particularly high between 2019 and 2020 (91.4%, Table 3).
Overall, the syphilis testing rate was 1.15 (95% CI, 1.12-1.19) per one person-year, in MSM the rate was 1.23 (95% CI, 1.19-1.26). The testing rate was lowest in 2018 (0.93, 95% CI, 0.87-1.00) and highest in 2021 (1.41, 95% CI, 1.34-1.48), however, the testing rate was similar in 2019 (1.14, 95% CI, 1.08-1.21) compared to 2020 (1.10, 95% CI, 1.04-1.16, P = 0.105 form the GEE model with Tukey-Kramer adjustment) (Fig. 2B). The proportion of MLWH who had at least one syphilis test per calendar year ranged from 77.7 to 87.9% and was 8.5% lower in 2020 compared to 2019 (Fig. 2B). The frequency of testing in the whole population and MSM is presented in more detail in Supplementary Tables 1S and 2S. Of 1187 men, 513 (43.2%) missed an annual syphilis test (Supplementary Table 3S). Those who had missed at least one annual syphilis test compared to those who had not were: older, acquired HIV more frequently by heterosexual transmission, had a longer history of ART, and more frequently lived outside Zagreb. Participants who had at least one annual syphilis test had more frequently past syphilis or syphilis at inclusion into the study (53 of 674) compared to those who did not have regular annual tests (28 of 513), but this difference was not statistically significant (P = 0.100, Supplementary Table 3S).
The number of syphilis events, follow-up time, events per 100 PYFU, and incidence rate ratios are shown in Table 2 and Supplementary Fig. S1. On crude analysis we observed evidence of a higher rate of a new episode of syphilis among MSM, those aged less than 40, those diagnosed with HIV and syphilis more recently, living in Zagreb and in MLWH who had past evidence of syphilis ( Table 2, Supplementary Fig. S1). Syphilis diagnoses per 100 person-years were similar in the age time-updated groups 18-29 years (N = 26, rate = 6.9 [95% CI, 4.7-10.1]) compared to the 30-49 years (N = 169, rate, 7.0 [95% CI, 6.0-8.1]) and higher compared to those older than 50 (N = 59, rate, 4.2 [95% CI, 3.2-5.4]). On multivariable analysis, many of the relationships found on crude analysis were less pronounced, statistical significance was found for MSM versus others, more recent calendar year of HIV diagnosis (2016-2020 versus ≤ 2010), more recent calendar years of syphilis diagnosis, and having a previous episode of syphilis (Table 2, Supplementary Fig. S1). A previous clinical AIDS diagnosis was associated with a decreased risk of having syphilis on follow-up (Table 2, Supplementary Fig. S1). The annual rate of asymptomatic and symptomatic syphilis diagnoses and the percent increase compared to the previous years are shown in Table 3.
We performed several other analyses which included only observations with at least one syphilis test done in a calendar year or included only MSM (Supplementary Tables 4S-6S and Supplementary Figs. S2-S4). In those analyses, the rates of syphilis events were higher than in our main analysis. The overall rate per 100 PYFU was as follows: 7.2 (95% CI, 6.3-8.1) in all men who had a syphilis test done at least once annually, 7.0 (95% CI, 6.2-8.0) in MSM, and 8.1 (95% CI, 7.1-9.2) in MSM with annual syphilis tests done.

Discussion
In this nationwide study of men living with HIV, the incidence of syphilis increased by 91.4% in 2020 compared to 2019, and this increase was mainly driven by an increase in symptomatic cases (106.1%) ( Table 3). We also found a 30.7% increase in the incidence of syphilis in 2021 compared to the previous year, but this increase was larger in asymptomatic syphilis cases. In our models, the incident rate of syphilis was at least > 2.5 times higher in 2021 compared to 2018. The increase in syphilis between 2020 and 2019 occurred in the background of a somewhat higher syphilis testing rate in 2019 (1.14 tests per one person-years) compared to 2020 (1.10 tests per one person-years).   www.nature.com/scientificreports/ The overall incidence rate of syphilis in our men living with HIV was 6.0/100 PYFU and the annual incidence rate ranged from 3.3/100 PYFU in 2018 to 9.3/100 PYFU in 2021. Similarly, to our findings in 2021, a retrospective cohort study from Australia reported an incidence of early syphilis of 9.  25 . Some studies reported somewhat lower incidence rates in PLWH than ours. A study from the HIV Swiss cohort reported data from October 2017 to November 2019 and found an incidence of syphilis of 4.4 cases per 100 person-years in male PLWH and of 6.3 cases per 100 person-years in MSM 26 . A recent analysis from the French Dat' AIDS HIV cohort including men and women, found an incidence of syphilis of 3.8 per 100 person-years for infection and 6.5 per 100 person-years for re-infection in the period 2010-2019 27 . Data from four US clinical sites reported an incidence rate of 5.6/100 PYFU in men and 6.9/100 PYFU in MSM in the period 2014-2018 28 . There are reports on an increasing number of cases of syphilis in some countries during the COVID-19 years 2020 and 2021 9-11 , however, data on the incidence density rates are lacking.
This huge increase in syphilis in MLWH occurred in Croatia one to two decades after similar outbreaks were reported in North America and Western Europe. Outbreaks of syphilis among MSM have been reported from the US since 2000 29 and ECDC reported a sharp increase (> 50%) of notifiable cases in several European countries (Germany, Denmark, Greece, Luxembourg, Malta, Norway, Portugal, and Sweden) between 2008 and 2013 30 . This increase in syphilis cases in Europe continued for at least up to 2019 19 . ECDC reported that 68% of syphilis cases were in MSM in 2019 and that among the countries that reported the HIV status 43% of cases were in PLWH 19 . It has been suggested that the high incidence of syphilis among MSM and PLWH could be due to risk perception, risky sexual behaviors such as unprotected anal sex, occasional sexual partners, concurrent sexual partners, HIV serosorting, chemex, transactional sex, methamphetamine use, group sex or contacting partners over the internet, and anonymous couples [31][32][33][34][35] . Closed sexual networks are important contributors to syphilis outbreaks 36,37 . As we have not collected data on sexual risks, we do not know whether those practices changed during the COVID-19 pandemic. However, lockdown measures and measures of social distancing with other precautions might have given a false sense of security. In Croatia, the sexual network dynamic may have changed during 2019 and 2020 leading to a local syphilis epidemic. There is little data on risk behaviors in PLWH from Croatia, however, a study conducted in 2005/2006 reported that 20% of MSM engaged in unprotected, non-concordant anal sex in the previous 6 months 38 . A recent study on HIV transmission clusters in newly diagnosed PLWH from Croatia found that 86% (347 of 403) belonged to an HIV cluster of which the largest included 53 persons 39 . We could expect that similar clusters of syphilis have occurred during the current outbreak.
Risk factors associated with syphilis such as being MSM and having a history of previous syphilis have also been reported previously 22,23,27,[40][41][42][43] . Younger age was also associated with syphilis; however, this relationship was not as strong as in other studies 40,43 . Interestingly, in the French Dat' AIDS cohort, age > 35 years was associated with a first or re-infection syphilis episode 28 whereas, in the recent study from the US, syphilis incidence was highest in the 30-39 age group 28 . Of note, having a past history of clinical AIDS was related to a reduced risk for syphilis, this was also found in the univariable but not multivariable analysis in the French Dat' AIDS cohort 27 . There was no association of detectable HIV-1 viral load and syphilis which implies that the risky sexual behavior leading to syphilis will not increase in HIV cases. Some recent studies found an association of HCV infection with syphilis 23,28 ; this was not the case in our study possibly because of a small number of HCV infections. Living in Zagreb the capital of Croatia was associated with syphilis mainly in our univariable analyses, syphilis outbreaks are especially common in large cities with significant populations of MSM 23,24,28,37,42 . The incidence of syphilis www.nature.com/scientificreports/ was higher in persons diagnosed with HIV in more recent years which might suggest that newer generations of MLWH have a different perception of risk and risky behavior. Similarly in a recent study from four US sites, a more recent entry into the cohort was associated with a higher incidence of syphilis 28 . There was overall an increasing trend in the annual syphilis serologic testing rate from 2018 to 2021, however, the rate was lower in 2020 by 3.5% compared to 2019. Similarly, the proportion of men who had at least one annual syphilis test was lower in 2020 compared to 2019. It seems that measures to curb the COVID-19 epidemic had a negative impact on syphilis testing, similar suggestions on sexual health screening and patients' referrals to STD clinics have also been made 44,45 . In Croatia, the first COVID-19 patient was documented on February 26, 2020, there have been several epidemic waves, and the highest number of confirmed cases was recorded in January and February 2021 (https:// covid 19. who. int/ region/ euro/ count ry/ hr, accessed Jun 7, 2023). A strict lockdown, including travel bans within the country, was implemented between March 19th and April 27th, 2020. This was followed by less strict measures (e.g. temporary closures of restaurants, bars, and fitness facilities, suspension of public events) that varied over time.
We have confirmed findings from others 46,47 that repeated episodes of syphilis are more frequently asymptomatic. However, it is still debated whether this observation is a result of an immune response to a previous infection or an increased frequency of serological testing 47 . We found an increased likelihood of early latent syphilis in PLWH with a repeated syphilis episode in the background of a similar testing rate.
Our study has limitations. This was a retrospective longitudinal observational study, so causal inferences cannot reliably be made. We did not collect data on specific sexual practices, condom use, or other risky behaviors such as chemsex, methamphetamine use, group sex, transactional sex, or use of dating sites to find new partners. Hence, we cannot report on the risky behaviors that contributed to the rise in syphilis cases. The calculation of syphilis incidence depends on the definition of syphilis and testing. Our definition of a syphilis event was not solely based on laboratory confirmation of the infection with both a treponemal and non-treponemal test. Eighteen events did not have a positive RPR test, 12 of whom were in persons with signs of a genital ulcer and 6 were in asymptomatic persons who had TPHA seroconversion. All had an epidemiological link to a syphilis case and all received treatment for syphilis. Hence, by the inclusion of those cases, we might have slightly overestimated the incidence of syphilis. However, we also might have underestimated the incidence of syphilis by not including syphilis episodes at inclusion into care and by including in the study only participants who had at least a 1-year follow-up. We also included in the analysis persons with no annual syphilis tests because we did not want to exclude those at lower risk for syphilis infection. Finally, we might not have captured all syphilis tests done in our study population because persons might have done them in other healthcare facilities. However, it is unlikely that many persons in HIV care received a syphilis test outside UHID, because of the stigma surrounding HIV infection, MSM, and STDs in general in Croatia. The strength of the study is that we provided national data on syphilis incidence in men living with HIV in Croatia and provided convincing evidence of a huge rise in syphilis between 2019 and 2020. All our additional analyses including men who had annual syphilis tests and only MSM confirmed our main findings (Supplementary Tables 4S-6S and Supplementary Figs. 2-4).

Conclusions
We report a 91% rise in the incidence of syphilis in men living with HIV in Croatia between 2019 and 2020. MSM were mainly affected. Traditional syphilis control strategies such as timely diagnosis and treatment, partner notification and treatment, and education/awareness campaigns might not be sufficient to curb the epidemic in men living with HIV We should strongly consider introducing doxycycline post-exposure prophylaxis which has been shown to significantly reduce syphilis in 3 randomized trials 48-50 . We should, however, also take into account that the wide use of doxycycline might increase antibacterial resistance and alter the microbiome, both of which are currently under investigation. Since many cases of syphilis are asymptomatic, serological screening should be increased particularly in those who had a previous syphilis episode. We showed that repeated episodes of syphilis are more frequently asymptomatic and not related to the frequency of testing. Longitudinal studies on the host humoral and cellular immune response in syphilis during index and repeated syphilis episodes could inform us about the underlying mechanisms of different clinical presentations.

Data availability
Data can be made available from the corresponding author upon reasonable request.